High LET Particle Therapy Targets Tumor Microtube Networks in Glioblastoma

Purpose: Tumor cell networks formed by tumor microtubes (TMs) are thought to drive therapy resistance in glioblastoma (GB). X–ray irradiation enhances TM formation, thereby increasing radioresistance. We hypothesize that high linear energy transfer (LET) particle radiotherapy is less affected by TM mediated resistance due to its reduced reliance on indirect DNA damage. This study explores the impact of LET-induced DNA damage on TMs formation and GB survival Material and Methods: Formation of TMs was investigated in the primary patient derived glioblastoma stem-like cell lines (S24 and T269) irradiated with different LET, ranging from 3 – 107 keV/μm, across dose series (1, 2, 4, 6 Gy) of clinical proton, helium, and carbon ion beams. TM networks and DNA damage patterns, specifically γH2AX foci, were visualized using fluorescence microscopy. Cell survival was evaluated through clonogenic survival assays. Results: The formation of TMs, radiation-induced nuclear DNA damage repair foci, and GB cell survival were correlated with a gradual increase in LET. Consistent with conventional photon/X–rays, low LET proton irradiation promoted TMs formation in a dose dependent manner. In contrast, an anticorrelation between LET and TMs induction was found, i.e., a decreased network connectivity with gradual increase of LET and formation of complex DNA damage. Consequently, LET increase correlated with reduced cell survival, with the most pronounced cell killing observed after high LET carbon irradiation. Moreover, the inverse correlation between LET and TMs density was further confirmed for a broad range of LET modulated within the carbon ion irradiation. Conclusion: This is the first report on the relevance of LET as a novel mean to overcome TMs network-mediated radioresistance in GB, with ramifications for the clinical translation of high LET particle radiotherapy to further improve outcome in this still devastating disease.